DESCRIPTION: The treatment of osteochondral defects (OCDs), which involve damage to both the subchondral bone and articular cartilage in the affected joint, is challenging. Such debilitating defects lead to mechanical instability, pain and worsening osteoarthritic degeneration. Current therapies fail to consistently repair and restore tissue function. Osteochondral tissue engineering technology utilizing biomaterials in combination with recruited and/or transplanted cells, and/or bioactive factors has emerged as a promising alternative approach. Human mesenchymal stem cells (hMSCs) are an attractive cell source as they can easily be isolated from bone marrow, expanded in culture without losing multipotency, and under appropriate conditions can differentiate into cells of the osteogenic and chondrogenic lineages. RNA interference (RNAi) is a powerful tool permitting inhibition of gene expression at the post-translational level by the targeted destruction of specific mRNA molecules, and has the potential to revolutionize the functional repair of damaged tissue by decreasing the expression of specific proteins that negatively impact healing processes or by altering stem cell differentiation pathways. Importantly, RNAi molecules have been identified that can promote the osteogenic and chondrogenic differentiation of hMSCs. However, effective delivery of RNAi molecules to target cells in vivo remains a significant challenge limiting its therapeutic potentia. We have engineered biopolymer hydrogels capable of locally delivering bioactive RNAi molecules with tailorable release profiles for delivery to surrounding and encapsulated cells, and these gels have been used to spatially and temporally control cell gene expression and fate. Therefore, the central hypothesis of this application is that the controlled spatial and temporal presentation of dual opposing RNAi molecule gradients in a biopolymer hydrogel will drive osteogenesis and chondrogenesis of encapsulated hMSCs in opposite directions to form osteochondral constructs that can promote the healing of OCDs. This will be addressed by the following specific aims: (1) Engineer biopolymer hydrogels with opposing concentration gradients of two different siRNAs for spatiotemporally controlled, sustained gene knockdown, (2) Deliver RNAi molecules that promote osteogenesis and chondrogenesis from biopolymer gradient hydrogels and investigate their capacity to spatially guide the osteogenic and chondrogenic differentiation of encapsulated hMSCs, (3) Develop opposing RNAi molecule gradient hydrogels with tailorable dimensions using microfluidic technology, and (4) Assess the ability of the hydrogel constructs containing hMSCs and opposing RNAi molecule gradients to drive osteogenesis and chondrogenesis in vivo upon implantation into a rabbit OCD model. This application aims to demonstrate the utility of a new tissue engineering approach for enhanced osteochondral tissue regeneration, which would have great clinical utility by improving the quality of life of patients suffering from OCDs.